# OPK-2,007-0;Y

BruggemannChemical U .S., hc. Subsidiary ofthe L. Bruggemann KG Germany 15 Reese Avenue - Suite 200 Newtown Square, PA 19073 " USA March 7, 2011 Via Overnight Delivery 

# Bruggemann Chemical 

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Document Control Office (DCO) (7407M) Office of Pollution Control and Toxics (OPPT) U .S. Environmental Protection Agency (EPA) EPAEast Building, Room E6428 (202-564-8930) 1201 Constitution Ave., NW Washington, D

.C. 20004-3302 Attn: TSCA Section 4 40 C .F .R . 799.5087(Docket # EPA-HQ-OPPT-2007-0531) ~ .3.cRe: Letter of Intent/Submission of Data in Response to TSCA Section 4Test Rule; Methanesulfinic acid, hydroxyl-, monosodium salt (CASRN 149-44-0) Dear Sir or Madam

: By this letter, BruggemannChemical U .S., Inc. (BruggemannChemical)1 is notifying the U .S. Environmental Protection Agency (EPA) of its intent to satisfy the testing requirements for Methanesulfinic acid, hydroxyl-, monosodium salt with CASRN 149- 44-0 (also known as "sodium hydroxymethanesulfinate" or "sodium formaldehyde sulfoxylate") specified in the Toxic Substances Control Act (TSCA) section 4test rule that appears in 40 C

.F .R. § 799.5087 (Test Rule) . According to the Test Rule, a "Bacterial Reverse Mutation Test (in vitro) : 40 CFR 799 .9510" must be conducted for the chemical substance. As documented in the enclosed files, we have recently conducted the prescribed testing for the substance in accordance with OECD Guideline 471 which is consistent with the referenced EPA method. As noted in the study report, the test itemis considered to be non-mutagenic in the bacterial reverse mutation test. Accordingly, we trust that through the provision ofthe enclosed study report and robust summary BruggemannChemical has fulfilled its obligations under the Test Rule. 1 BruggemannChemical U .S., Inc. is located at 15 Reese Avenue, Suite 200, Newtown Square, PA 19073, telephone: (610) 353-6840. BriiggemannChemical U .S., Inc . is the U

.S. subsidiary of BrtiggemannChemical which is headquartered in Germany. 

## CONTAINS NOCEjt

a _ Edward 1. McDade, III For Deposit of LISD : Vice President Wachovia Bank Account: 2100012829009 ABA : 031201467 0 9001/14001 Swift Code: TNBTUS33 REG

. NR. 1499-02 For Deposit of EUR Baden-Wurttembergische Bank in Heilbronn (BLZ 60050101) i .e. Account #1430019 ABA : DE066 050 101 00014 300 19 Swift Code: SOLADE ST Will

Phone 610353 9852 Fax 610353 9853 info aClbrueggeman.com www .brueggemann .comShould you have any questions regarding this matter, or desire further information, please

do not hesitate to contact Mr . Ed McDade at (610) 353-9852 or Ed.McDade@brueggemann.com . Sincerely, Mr. David E. Wilson President

Enclosures 1 . Final Report - Determination ofthe mutagenic potential of NHS (Sodium hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B 13/14 . 2 . IUCLID Endpoint Study Record: Genetic toxicity in vitro.001 .,*nr~ , ~ r~eE, jr

Final Report

Determination of the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 

### IT 4 4

07120702G803Final Report of LAUS GmbH No.: AB07120702G803 Auf der Schafweide 20, D-87489 Kirrweiler Tel . ++49-8321-96299-0, Fax 98299-29, e-mail : infoClaus .de Date: 02. May 2008



### Final Report ORIGINAL

. Z OF _4Z Title : Determination of the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13114 Project No. : 07120702G803 Sponsor : BrUggemann Chemical UM Salzstraf3e 131 D-74076 Heilbronn Monitor : Dr. Franz NGRlein Test Item

: NHS (Sodium hydroxymethanesull "inate) Batch No. : 07041205 Test Facility: LAUS GmbH Auf der Schafweide 20 D-67489 Kirrweiler 

### Study Director: Dipl . Chem . Jbrg Paufus Deputy Study Director: Dipl . Ing . Anette Rudolf Time Table: Study Plan dated: Experimental Starting Date: Experimental Completion Date: Draft Report : Final Report : 08. Jan. 2008 16. Jan. 2008 25. Jan. 2008 29 . Jan. 2008 02. May 2008Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kircrweiler No. : AB07120702G803 Tel . ++49-8321-98299-0, Fax 96299-29, e-mail : info®Iaus .de f Date: 02. May 2008 This page was intentionally left blank for statement of sponsor or submitter.

page 2 of 37Final Report of LAUS GmbH Adder Schalwaide 20, D-67489 Kirrweiler No. : AB07120702G803 Tel . ++49-6321-96299-0, Fax 96299-29. e-mail : infoQlaus.de I Date: 02 . May 2008 I GLP-COMPLIANCE CLAIM It is hereby declared that all tests were made in accordance with the Revised OECD Principles of Good Laboratory Practice" (Paris, 1997) as stated in the following guidelines: OECD Principles of Good Laboratory Practice, adopted by Council on 26th November 1997; Environment Directorate, Organisation for Economic Cooperation and Development, Paris 1998 Directive 2004/10/EC of the European Parliament and of the Council of 11 February 2004 on the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances (codified version) ChemikaliengesetZ (Chemicals Act) of the Federal Republic of Germany (ChemG) §19 and annexes 1 and 2 in the version of 25. July 1994 (BGBI . I p. 1703), amendments as of 08

. May 2001 (BGBI . I, p. 843) and 20 . Jun. 2002 (BGBI

. I pp. 2090- 2130) . There were no circumstances that may have affected the quality or integrity of the study. This report contains data which were not determined under GLP conditions : Cytotoxicity (see annex 4) of the test itemwas determined before initiation of the study. Dipl . ~h,6m

."Jdi`g Study 

### G ~ 1.; ;,Y 2008 Date Personnel involved in the performance of the study: Isabell Andres Technical Assistant Biology Anette Rudolf Dipl .-Ing. Environmental Protection JUrgen Schlinck Technical Assistant Biology

page 3 of 37Final Report of LAUS GmbH Auf der schalWeide 20, 0-67489 Kirrweifer No. : AB07120702G803 Tel . *+49-6321-96299-0, Fax 96299-29, e-mail : infoClaus .de I Date: 02. May 2008 2 QUAL1Ty ASSURANCE STATEMENT Title: Determination of the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 Test Item

: NHS (Sodiumhydroxymethanesulfinate) Batch No. : 07041205 Project No. : 07120702G803 This study has been inspected by the quality assurance unit according to the principles of Good Laboratory Practice . Study Plan and Final Report were checked at the dates given below, the Study Director and the management were informed with the corresponding report . Also, the performance of the study was inspected, and findings were reported to Study Director and management . The inspection of short-term studies (duration less than one week) is carried out as audit of process concerning major technical phases of at least one similar test . Frequency is once or more a quarter. The study was conducted and the reports were written in accordance with the Study Plan and the Standard Operating Procedures of the test facility. Deviations from the Study Plan were acknowledged and assessed by the Study Director and included in the Final Report . The reported results reflect the raw data of the study. 

### Verified procedure Date of Inspection Audit report no . Audit report dated Study plan 09 . Jan . 2008 080109-01 09. Jan . 2008 Performance of study 23. Jan. 2008 080123-02 23. Jan. 2008 Draft report 07. Feb. 2008 080207-01 07. Feb. 2008 Final report 01 . May 2008 080501-03 01 . May 2008 

### ~' % , .r; ' 2008 Jutta Paulus Date Quality Assurance

page 4 of 37Final Report of LAUS GmbH No . : AB07120702G803 Auf der Schafweide 20, D-67489 Kinweiler Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus .de Date: 02. May 2008



### Table of Contents 1 GLP-COMPLIANCE CLAIM 3 2 QUALITY ASSURANCE STATEMENT 4 3 SUMMARY g 4 PURPOSE OF THE STUDY 10 5 LITERATURE 10 6 MATERIALS AND METHODS 10 6.1 Test Item 10 6.1 .1 Specification 10 6.1 .2 Storage 11 6.1 .3 Preparation 11 6.2 Reference Items 11 6.2.1 NPD 11 6.2 .2 Sodiumazide 11 6.2 .3 BaP 11 6.2 .4 2-AA 11 6.3 Test System 11 6.3 .1 Specification 11 6.3 .2 Origin and Culture 11 6.4 Chemicals 12 6.4,1 Nutrient broth for overnight culture 12 6,4.2 Isotonic sodiumchloride solution for dilution purposes 12 6.4 .3 Vogel-Bonner-Medium20fold 12 6.4 .4 Glucose solution 40% 12 6.4 .5 Minimal Glucose Agar 12 6.4 .6 Biotin Agar 12 6.4 .7 Histidine-Biotin-Agar 12 6.4 .8 Ampicillin-Agar 12 6.4 .9 Ampicillin-tetracycline plates 12 6.4 .10 Nutrient agar plates 13 6.4 .11 Basis for Top-Agar and Maximal-Soft-Agar 13 6.4 .12 Histidine-Biotin-solution 0.510.5 millim . (use: top agar) 13 6.4 .13 Histidine-Biotin-solution 5 millim .l 0.5 millim

. (use: maximal-soft-agar) 13 6.4 .14 Phosphate buffer 13 6.4 .15 Salt solution for S9-Mix 13 6.4 .16 NADP-solution for S9-Mix 13 6.4 .17 Glucose-6-phosphate solution for S9-Mix 13 6.4 .18 S9-Mix 13 6.4 .19 S9 13 6.5 Test Vessels 15 page 5of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kirrweiler Tel, ++49-6321-96299-0, Fax 96299-29, e-mail : infog 6.6 Instruments and Devices 7 PERFORMANCE OF THE STUDY 7.1 Culture of Bacteria 7.2 Description of the Method 7 .2.1 Preparations 7 .2.2 Experimental Parameters 7 .2.3 Description of the Method 

### 7.3 References 7 .3.1 Genotype Confirmation 7 .3.2 Determination of Titre 7 .3.3 Toxicity Control 7 .3.4 Sterility Control 7 .3.5 Positive Controls 7.4 Evaluation 8 FINDINGS 8.1 First Experiment 8.1 .1 Confirmation of the criteria 8.1 .2 Mutagenicity 8.1 .3 Survey of the Findings 8.2 Second Experiment 8,2.1 Confirmation of the Criteria 8.2 .2 Solubility and Toxicity 8.2 .3 Mutagenicity 8.2 .4 Survey of the findings 

### 9 RESULTS 10 DISCUSSION 11 DEVIATIONS 11 .1 Deviations fromthe Study Plan 11 .2 Deviations fromthe Guideline 12 RECORDING 13 ANNEX 1 : COPY OF GLP CERTIFICATE 14 ANNEX 2: DATA OF THE FIRST EXPERIMENT 14 .1 Determination of Titre 14 .2 Sterility Control 14 .3 Spontaneous Revertants 14.3.1 Water 14.3.2 DMSO 14 .4 Positive Controls laus .de No . : AB07120702G803 Date : 02. May 2008

15 16 16 16 16 16 17 18 18 18 18 18 19 19 20 20 20 20 21 22 22 22 22 22 23 23 23 23 23 23 24 25 25 25 26 26 26 27 page 6 of 3714

.6 Test Item NHS (Sodium hydroxymethanesuiffnate) 14.5.1 Mutagenicity Test 15 ANNEX 3

: DATAOF THE SECOND EXPERIMENT 15.1 Determination of Titre 15 .2 Sterility Control 15.3 Spontaneous Revertants 15.3 .1 Water 15.3 .2 DMSO 15 .4 Positive Controls 15 .5 Test Item NHS (Sodium hydroxymethanesulflnate) 15.5.1 Mutagenicity Test 16 ANNEX 4

: DATA OF THE PRE-TEST (CYTOTOXICITY) 16.1 Experimental Parameters 16.2 Determination of Titre 16.3 Toxicity Control 16.4 Assessment 17 ANNEX 5: COMPARISONWITH HISTORICAL DATA 18 ANNEX 6

: GENOTYPE CONFIRMATION 18.1 Histidine Requirement 18.2 Ampicillin-(Tetracyclinee)-Resist (pKM 101, pAQ1) 18.3 UV-Sensitivity (uvrB) 18.4 Crystal Violet Sensitivity (deep roughlrfa) 28 28 30 30 30 31 31 31 32 33 33 34 34 34 35 35 36 37 37 37 37 373 SUMMARY Final Report of LAUS GmbH Auf der Schafweide 20, D-B7489 Kirrweiler No . : AB07120702G803 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus .de I Date: 02. May 2008

Study: Determination of the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 Sponsor : Brtiggemann Chemical UM Salzstraf3e 131 D-74076 Heiibronn Monitor: Dr. Franz Nuf3lein Test Facility: LAUS GmbH Auf der Schafweide 20 D-67489 Kirrweiler Project No. : 07120702G803 Study Plan No. : PP07120702G803 Findings: Two valid experiments were performed. First Experiment : Five concentrations of the test item, dissolved in deionised water (ranging from 5047 to 51 pg/plate) were used . Five genetically manipulated strains of Salmonella fyphimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method . None of the concentrations caused an increase in the number of revertant colonies in the tested strains . The test itemdidn't show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn't show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. page 8 of 37Second Experiment :

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging from 5031 to 1258 irg/plate) and a modification in study performance (pre-incubation method) . The test itemdidn't showmutagenic effects in the second experiment, either . No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn't show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Under the conditions of the test, the test item didn't showmutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. Therefore, no concentration-effect relationship could be determined .' The test item NMS (Sodium hydroxymethanesulfinate) is considered as "not mutagenic under the conditions of the test" .

page 9 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-87489 Kirrweiler No. : AB07120702G803 Tel . ++49-8321-96299-0, Fax 96299-29, e-mail : itifo@laus .de I Date: 02. May 2008 4 PURPOSE OF THE STUDY This study was performed in order to evaluate the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) . The test was chosen on behalf of the sponsor for registration purposes following Chemicals Law . 5 LITERATURE The study was conducted in accordance with the following guidelines: OECD Guidelines for the Testing of Chemicals Bacterial Reverse Mutation Test" Part 471, adopted July 218t, 1997 EU-Guideline 671548 EWG, last changed by 29th amendment, Annex V, Method B.13/14 "Mutagenicity - Salmonella typhimurium, reverse mutation assay", adopted May 19th, 2000 Corresponding SOP of LAUS GmbH SOP 118 008 03 edition 6, dated 13. Nov. 2006 Bestimmung des erbgutverandernden Potentials mit demBacterial-ReverseMutation-Test (AMES-Test)" For historic data, the following literature was included: Revised Methods for the Salmonella mutagenicity test" by Dorothy Maron and Bruce Ames, Mutation Research 113 (1983), S. 173 - 215 6 MATERIALS AND METHODS 6.1 Test Item Prof.

6.1 .1 Specification The following information concerning identity and composition of the test item were vided by the sponsor . Name Batch no Appearance Composition

CAS-No. EINECS-No. Molecular formula Molecular weight Purity NHS (Sodiumhydroxymethanesulfinate) 07041205 white powder main component Sodiumhydroxymethanesulfinate 4.4 - 6 %Na-hydroxymethansulfonat 1 .9 - 5%Na-sulfit 2.4 - 3%Na-sulfat 1 .2 - 2 %Na-carbonat 11 .5 -15.5 %(crystal) water 149-44-0 2057394 CH303S"Na resp. CH303S*Na x 2 H20 154.1 glmol 99.3%(dihydrate), 76.1% (anhydrous) propage 10 of 37Homogeneity

Volatility Stability Solubility Date of+production Date of expiry Storage conditions Hazard information R-phrases S-phrases Final Report of LAUS GmbH Auf der Schalweide 20, D-67489 Kirrvveiler No . : AB0712070213803 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus .de I Date: 02. May 2008 homogeneous not volatile H20 96h, EtOH, acetone, CH3CN, DMSOnot stated H20 >1gIL EtOH, acetone, CH3CN, DMSOnot stated 12. Apr. 2007 12. Apr . 2009 roomtemperature 20 t 5 °C none none none 6.1 .2 Storage The test itemwas stored in a closed vessel at room temperature . 6.1,3 Preparation Preparation was the same for both experiments . On the day of the start of the experiment, a stock solution containing 50 gIL of the test item in deionised water was prepared . Water was chosen as solvent, because the test item was completely soluble, and this solvent doesn't have any effects on the viability of the bacteria or the number of spontaneous revertants. The stock solution was used to prepare the geometric series of the concentrations to be tested . Each solution was membrane 5ltrated to accomplish sterility. 6.2 Reference Items The following mutagenic substances were used as positive controls: 6.2.1 NPD 4-Nitro-1,2-phenylene diamine, C6H7N302; CAS-No. : 99-56-9 6.2 .2 Sodiumazide Sodiumazide, NaN3; CAS-Nr. : 26628-22-8 6.2 .3 BaP Benzo-a-pyrene, C2oH12; CAS-Nr . : 50-32-8 6.2 .4 2-AA 2-Amino-anthracene, C14H11N; CAS-Nr. : 613-13-8 6.3 Test System 6.3 .1 Specification Species Salmonella typhimurium LT2 Strains TA 1535, TA 97a, TA 98, TA 100 and TA 102 Mutations TA97a: hisD6610, TA 98: hisD3052 TA 100 und TA 1535 hisG46, TA102:

hisG428, TA97, TA98 and TA100 contain uvr8 ; TA97, TA98, TA100 and TA102 contain pKM

101 and rfa. 6.3.2 Origin and Culture Salmonella typhimurium (all strains used) were obtained from Prof . Ames (date of arrival : Feb. 25th 2002 (TA97a, 98, 102), June 23`d 2001 (TA 100) and Jan. 29°h, 2001 (TA1535)) and stored as stock cultures at - 80°C . One day before the start of each experiment, one vial per strain to be used was thawed and put into a culture vessel containing nutrient page 11 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kirrweiler No. : AB07120702G803 Tei, ++49-6321-96299-0, Fax 96299-29, e-mail : infoClaus .de I Date: 02 . May 2008 broth. After incubation over night at 37 C, the cultures were used in the experiment . During the test, the cultures were stored at roomtemperature as to prevent changes in the titre . 6.4 Chemicals The purity of the chemicals which were used were either analytical grade" or for microbiological purposes" . Every substance was controlled upon arrival in the test facility. All solutions and media were sterilized, either by autoclaving (121 ~C, 20 minutes) or by membrane filtration . The weights may differ fromthe theoretical value (difference < 5 %) . Additionally, differing specifications (e.g . water of crystallization) may be used. 6.4.1 Nutrient broth for overnight culture Nutrient broth Merck 5443 2.8 g H20 demin . ad 350 mL 6.4.2 Isotonic sodiumchloride solution for dilution purposes Sodiumchloride 0.9 g HZO demin . ad 100 mL 6.4.3 Vogel-Bonner-Medium20fold Magnesiumsulphate (MgS04''7H20) 4.0 g Citric acid mono hydrate 40.0 g Potassium phosphate, dibasic (anhydrous) (K2HP04) 200.0 g Sodiumammoniumphosphate, monobasic, tetra hydrate (Na(NH4)HP04*4H20) 70.0 g H20 demin. ad 1000.0 mL 6.4 .4 Glucose solution 40% Glucose 400.0 g H20 demin. ad 1000.0 mL 6.4 .5 Minimal Glucose Agar Vogel-Bonner-Solution 20fold 500.0 mL Glucose solution 40% 500.0 mL H20 demin . 9000.0 mL Agar 150.0 g 6.4.6 Biotin Agar

. Minimal-Glucose-Agar, 80 C 500.0 mL Biotin solution 0.5 millimolar 3.0 mL 6.4.7 Histidine;Biotin-Agar Biotin-Agar, 80 C 350.0 mL Histidine solution 0.5% 3.5 mL 6.4 .8 Ampicillin-Agar Histidine-biotin agar, 80 °C 250.0 mL Ampicillin solution 0.8% 0.6 mL 6.4 .9 Ampicillin-tetracycline plates Ampicillin agar, 80 C 50.0 mL Tetracycline solution 0.8% 0 .01 mL page 12 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kirrweiler No. : AB07120702G803 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus .de I Date: 02. May 2008 6.4 .10 Nutrient agar plates Nutrient broth Merck 5443 0.8 g Sodiumchloride (NaCl) 0.5 g Agar 1 .52 g HZO demin. 100 mL 6.4.11 Basis for Top-Agar and Maximal-Soft-Agar Agar 6 g Sodiumchloride (NaCl) 5 g H20 demin . ad 1000 mL 6.4.12 Histidine-Biotin-solution 0.5/0.5 millim . (use: top agar) D-Biotin (MG 247.3) 12.36 m 9 L-Histidine* HCI"`1 H20 (MG 209.7) 10.5 mg H20 demin ., 90°C ad 100.0 mL To 100 mL basis (see 7.4 .11), 10 mL histidine-biotin-solution 0.5/0.5 millim . are added. 6.4.13 Histidine-Biotin-solution 5 millim ./ 0.5 millim

. (use: maximal-soft-agar) D-Biotin (MG 247.3) 12.36 mg L-Histidine"' HCI*1 H20 (MG 209.7) 105 mg H20 demin., 90°C ad 100.0 mL To 100 mL basis (see 7.4 .11),10 mL histidine-biotin-solution 5/0.5 millim . are added . 6.4 .14 Phosphate buffer Sodiumdi-hydrogen phosphate monohydrate NaH2P04*H20 0.3312 g Di-sodiumhydrogen phosphate NaZHP04 2.5 g H20 demin . ad 100.0 mL The pH of the solution is adjusted to 7 .4 t 0 .1 . 6.4 .15 Salt solution for S9-Mix Potassiumchloride (KCI) 1 .23 g Magnesiumchloride hexahydrate MgCl2'`6H20 0.814 g H20 demin. ad 10.0 mL 6.4 .16 NADP-solution for S9-Mix NADP (MR = 765.4 g/mol) 766 mg H20 demin. 10 mL 6.4 .17 Glucose-6-phosphate solution for S9-Mix Glucose-6-phosphate (MR = 282 g/mol) 564 mg H20 demin., sterile ad 2 mL 6.4 .18 S9-Mix Phosphate buffer 22,5 mL 0.1m NADP-solution 1 .0 mL 1 m-G6P-so[ution 0.125 mL Salt solution 0 .5 mL Rat liver S9, 4°!0 1 .0 mL 6.4 .19S9 S9 was obtained by Trinova Biochem, Gief3en . Batch no: 2118, 2081, 2113 page 13 of 37Final Report of IJAUS GmbH Auf der Schafweide 20, D-67489 Kirrvveiler No. : AB07120702G803 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus .de I Date : 02. May 2008 Specification : produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.

page 14 of 37Final Report of LAUS GmbH Aut der Schatweide 20, D-67489 Kirrvreiler Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info laus.de No. : AB07120702G803 Date: 02. May 2008 6.5 Test Vessels All vessels used are made of glass or sterilizable plastic. They were sterilised before use by heating to 180 °C (two hours) or autoclaving. The following vessels were used : Schott-bottles, glass vials, and culture flasks for solutions and media Plastic petri plates Glass tubes (with Kapsenberg caps) for TopAgar-bacteria-substance mix 6.6 Instruments and Devices The following instruments and devices were used in the performance of the study. Autoclave MLS 3020 Magnetic stirrer and magnetic stir bars Scales Mettler PB 5001-S02 Analytical scales MT AB 184 SA Incubation chamber Heraeus, adjustable to 37 °C Table water bath GFL, adjustable to 45 °C Heating chamber Memmert 1 Orbital shaker GFL 3005 Adjustable pipettes with sterile tips, LAUS No. 8 (0,2-2ml), LAUS No. 22 (20-200p1), LAUS No. 32-33 (500N1), LAUS No. 34-38 (100N1) Glass thermometer LAUS No. 20050117-74; 20020912-22; 20020912-15 Glass measuring cylinders, 100 and 1000 mL Glass measuring flasks, 25, 500 and 1000 mL Membrane filters, 0.2 pm pore diameter Drigalski spatula Usage and, if applicable, calibration following the corresponding SOP in the current edition.

page 15 of 37Final Report of LAUS GmbH Auf der Schatweide 20, D-67489 Kirrweiler Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info(p3laus .de 7 PERFORMANCE OF THE STUDY 7.1 Culture of Bacteria No. : AB07120702G803 Date: 02. May 2008

12 hours before the start of each experiment, a stock culture of each strain was thawed and an aliquot was placed into a culture flask containing 70 mL nutrient broth. The flasks were incubated at 37°C for 12 hours . 7.2 Description of the Method 7 .2.1 Preparations In the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilised and the first batches poured. On the day before the test, the remaining plates were poured. On the day of the test, the overnight cultures were checked for growth. The incubation chamber was heated to 37 °C . The water bath and the heating block were turned to 43 °C. The table surface was disinfected . The S9 mix was freshly prepared and 7 .2.2 Experimental Parameters 7.2.2 .1 First Experiment Date of treatment Concentrations tested Tester strains Incubation time Incubation temperature Tester strains Method References 7.2 .2 .2 Second Experiment Date of treatment Concentrations tested Tester strains Incubation time Incubation temperature Method References stored at 0 °C . January 161h, 2008 5047 1 1514/5051151 /51 Ng/plate TA97a, TA98, TA100, TA102, TA 1535 48 hours 37 °C TA97a, TA98, TA100, TA102, TA1535 plate incorporation method see under 7.3.5 January 23`4, 2008 5031 I 2516 I 1258 pg/plate TA97a, TA98, TA100, TA102, TA1535 48 hours 370C pre-incubation method see under 7.3.5

page 16 of 37Final Report of LAUS GmbH Auf der Schafwelde 20, D-87489 Kirrweiler Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info 7 .2.3 Description of the Method laus .de No . : AB07120702G803 Date : 02. May 2008

7 .2.3.1 Plate incorporation method Per strain and dose, four plates with and four plates without S9 mix were used . 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels . Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C. 0.1 mL of the appropriate solution of the test itemwere given into a sterile tube . After mix- ing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added . The mixture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few

minutes, then inverted and placed in the dark incubator at 37 °C . 7.2.3.2 Pre-incubation method Per strain and dose, four plates with and four plates without S9 mix were used . 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine- biotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C. 0 .1 mL of the appropriate solution of the test itemwere given into a sterile tube. After mix- ing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking . Then 2 mL top agar were added. The mixture was vortexed gen- tly, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula . The plates were closed, covered with brown paper and left to harden for a fewmin- utes, then inverted and placed in the dark incubator at 37 °C.

page 17 of 37

### 7.3 References Final Report of LAUS GmbH Auf der Schafweide 20, D-87489 Kirroreiler No. : AB07120702G803 Tel, ++49-8321-96299-0, Fax 96299-29, e-mail : infoQlaus.de I Date: 02 . May 2008

7.3 .1 Genotype Confirmation Genotype confirmation is performed once a term . 7.3 .1 .1 Histidine requirement Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilised wire loop. 7.3 .1 .2 Ampicillin-Resistance (pKM101) resp. ampicillin-tetracycline-resistance (pAQ1) The strains were streaked on ampicillin agar, TA102 on ampicillin-tetracycline agar . TA1535 was taking the function of control strain, since it is not ampicillin resistant . 7.3 .1 .3 UV-sensitivity (uvr8) Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light . The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 33 cm . Incubation over night at 37 °C followed . 7 .3 .1 .4 Crystal violet sensitivity (deep rough) For each strain, two plates were used . 0 .1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (Q1 9 mm), each soaked with 10 NI of crystal violet solution (0.1%) were placed into the middle of each plate, fol- lowed by incubation over night . 7 .3.1 .5 Spontaneous revertants Four replicates, with/without S9, for each solvent which was used in the test . 7 .3.2 Determination of Titre The titre was determined by dilution of the overnight culture using sodiumchloride solution and placing 0.1 mL on maximal-soft agar. It should give a density of 109 ceIIsImL (at the least) . 7.3.3 Toxicity Control Performed analogously to the titre control with the maximumdose of test item with and without S9 on maximal-soft agar. 7.3.4 Sterility Control Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar .

page 18 of 37

### Final Report of LAUS GmbH Auf der Schaiweide 20, D-67489 Kirrweiler Tel, i+49-8321-98299-0, Fax 96299-29, e-mail : infoClaus.de No. : AB07120702G803 Date: 02. May 2008 7.3 .5 Positive Controls Using diagnostic mutagens, four replicates were prepared . The stock solutions of the sub- stances were diluted to effect an application volume of 0 .1 mUplate. 7.3 .5.1 Without metabolic activation system + 4-Nitro-1,2-pheny(ene diamine Concentration per plate: 80 Ng; strains TA 97a, TA 98 and TA 102. + Sodiumazide Concentration per plate: 6 Ng; strains TA 100 and TA 1535. 7 .3.5.2 With metabolic activation system + Benzo(a)pyrene Concentration per plate: 40 Ng; strain TA 98. 2-Aminoanthracene (2-AA) Concentration per plate: 3 pg ; strains TA 97a, TA 100, TA 102 and TA 1535. 7.4 Evaluation The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excelo) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction . A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >_ 2) in at least one strain can be observed . A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

page 19 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kinvreiler Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@Isus.de 8 FINDINGS The detailed data are listed in the annex. 8 .1 First Experiment No. : AB07120702G803 Date: 02. May 2008

8.1 .1 Confirmation of the criteria The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see page 36) . All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation. The test itemwas dissolved in deionised H20, preparing a stock solution containing 50gIL (nom .) . No signs of toxicity towards the tested strains could be observed : the background lawn was visible, the number of revertant colonies was not significantly reduced . 8.1 .2 Mutagenicity No significant increase of the number of revertant colonies in the treatments with and with- out metabolic activation could be observed . No concentration-related increase over the tested range was found . Therefore the test item is stated as not mutagenic under the test conditions. To verify this result, a second experiment was performed using the pre-incubation method.

page 20 of 378.1 .3 Survey of the Findings

The mean revertant values of the four replicates are presented in the following table. Table 8.1-a Mean Revertants First Experiment Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 H 0 Mean 131 96 10 9 190 161 171 180 10 12 2 sd 49 4 5 4 33 26 36' 72 1 3 DMSO Mean 116 111 12 9 180 182 178 172 14 21

sd 42 40 3 1 28 20 10~ 20 3 10 Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Pos . Contr . sd 0 0 0 0 0 0 0 0 0 0

f(l) 8.63 9

.02 83.42 111 .2 5.27 5 .50 5 .62 5.82 100

.1 47 .67 Mean 119 110 12 7 164 140 82 157 10 16 5047 pg/pl . sd 47 16 5 2 38 14 24 33 4 3

f(l) 0 .91 1 .15 1 .20 0

.78 0.86 0

.87 0 .48 0 .87 1 .00 1 .33 Mean 105 137 11 7 182 163 152 136 10 8 1514 pg/pl . sd 23 23 2 5 39 46 46 57 1 1

f(l ) 0 .80 1 .43 1 .10 0

.78 0.96 1 .01 0

.89 0 .76 1 .00 0

.67 Mean 136 122 10 8 168 143 125 117 9 10 505 pg/pl . sd 32 41 3 2 __ 28 24 14 20 1 1

f(l) 1 .04 1 .27 1 .00 0 .89 0 .88 0 .89 0 .73 0 .65 0 .90 0.83 Mean 98 123 10 10 134 176 130 132 11 11 151 pg/pl . sd 40 20 2 _ 2 43 36 18 18 5 4

f(l) 0 .75 1 .28 1 .00 1 .11 0 .71 1 .09 0

.76 0 .73 1 .10 0.92 Mean 119 139 12 6 193 167 170 149 14 9 511aglpl . sd 30 22 5 _ 1 31 27 32 31 4 1 f( l)l 0 .91 ~ 1 .45 1 1 .20 0

.67 1 .02 ~ 1 .04 0

. 99 0 . 83 1 .40 0

. 75 In this table, "1001" represents "> 1000" .

page 21 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kirrvveiler Tel . ++49-6321-96299-0, Fax 98299-29, e-mail : info 8.2 Second Experiment laus .de No. : AB07120702G803 Date: 02. May 2008

8.2.1 Confirmation of the Criteria The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range -of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation . 8.2.2 Solubility and Toxicity The test itemwas dissolved in deionised H20, preparing a stock solution containing 50 g/L (nom .) . No signs of toxicity towards the tested strains could be observed : the background lawn was visible, the number of revertant colonies was not significantly reduced . 8.2.3 Mutagenicity No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed . No concentration-related increase over the tested range was found. Therefore the test item is stated as not mutagenic under the test conditions. 8.2.4 Survey of the findings The mean revertant values of the four replicates are presented in the following table. Table 8 .2-a Mean Revertants Second Experiment Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 ~ +S9 -S9 +S9 H 0 Mean 122 175 12 10 116 124 163 238 10 11 2 sd 35 14 3 6 9 14 35~ 29 4 3 DMSO Mean 137 154 16 10 _ 150 148 192 208 13 12 sd 23 24 6 2 46 37 45 63 1 4 Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Pos .Contr . sd 0 0 0 0 0 0 0 0 0 0

f(l) 7.31 6

.50 62.56 100

.1 8.63 6

.76 5.21 4

.81 100 .1 83.42 Mean 126 154 10 12 143 147 185 240 14 11 5031 Nglpl . sd 14 18 2 4 12 50 46 41 3 3

f(l) 1 .03 0

.88 0 .83 1 .20 1 .23 1 .19 1 .13 1 .01 1 .40 1 .00 Mean 102 101 12 10 130 _ 153 236 188 9 15 2516 pg/pl . sd 26 25 5 1 15 52 32 56 2 2

f(l) 0 .84 0 .58 1 .00 1 .00 1,12 1 .23 1 .45 0 .79 0 .90 1 .36 Mean 140 129 11 10 203 151 228 168 11 13 1258 Nglpl . sd 57 33 4 5 8 30 13 37 2 0

f(U ~ 1 .15 1 0

.74 1 0 .92 1 1 .00 1 .75 1 .22 1 .40 0 . 71 1 . 10 1 . 18 In this table, "1001" represents "> 1000" .

page 22 of 379 RESULTS

The test item didn't showmutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only) . Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased . Therefore it can be stated, that under the test conditions, the test item NHS (Sodium hydroxymethanesulfinate) is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurlum, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. 

### 14 DISCUSSION The test itemis considered not mutagenic for the reasons given above. Also, the test item didn't show any cytotoxicity towards the bacteria. The confirmation tests of the genotype didn't showany irregularities . The control of the titre was above the demanded value. The positive controls didn't evoke as many mutations as given by Prof. Ames . But the number of revertant colonies was in the range of the historical data of the laboratory (see page 36) and were definitely increased in comparison with the negative controls, as well as showing mutagenous potential of the diagnostic mutagenes . Some of the spontaneous revertants are lower than the values given by Prof . Ames; in comparison with the historical data of the LAUS GmbH, they were within the normal range . For these reasons, the result of the test is considered valid . 11 DEVIATIONS 11 .1 Deviations fromthe Study Plan No deviations fromthe study plan were observed . 19.2 Deviations fromthe Guideline None as known . 12 RECORDING One original of study plan and final report, respectively, all rawdata of the study and all documents mentioned or referred to in study plan or final report will be kept in the GLP Document Archive of the test facility for fifteen years. After that, the sponsor's instructions will be applied (shipment of documentation to sponsor) . A retain sample of the test item will be kept in the GLP Substance Archive for fifteen years; then, the retain sample will be discarded. Number of originals which will be sent to the sponsor: 1

page 23 of 37p. 27

Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kirrvveiier Tel . t+49-8321-96299-0, Fax 96299-29, e-mail : infoQiaus .de 13 ANNEX 1 : COPY OF GLP CERTIFICATE

Gute Laborpnxis I Good Laboratory Practice No . : AB07120702G803 Date: 02. May 2008

UP-Bescheinigung I Statement of GLPCompliance

(gem . /according to § 19 Aba. 1 Chemika8engesetz) Em* GLP-Inapektlon zur Uberwachung der F-arhattung der GLP-Grundeatze qemar6 Chemikakenpesetz bzw

. Richtlime 88I320IEG wurde durohgetuhrt in Asaessrnant of conformity with GLP according lo Chemikakengesetz and Directive ttal340l EEC at PrUfeinrichtung I Test facility LAUS GmbH Auf der Schafweide 20 67489 Kirrevatler PrUfung nach Kete(iiorien 1Areas of Expertise (psm Iaooordwg CMmVwV-oLP Nr S3t0ECD qudants)

1, 3 .4, 6, 6, 8

Theabaft maniaHd taqtacky p bRprdad In The nopOnsl GLP Caeo4na Pwpramma a n d is Inspected on a npular haut aasW on Ors Inpadlon rarwrr M can be eon-IIYmad, trut ft reat rapfirY o ate t0 ¢enduW4 tM arorem0alqned stunnin wmprlana vrin ft Principlas at GLP Varft0tbn of the oomPUance or the tog fatddy wth Re Ptit4a<a or the GLP

has to be appaed for in UnM t0 atbw for a rapow

"rq wtapH,y,pn to take OW wRno bog years Me, rarnananp" Vhf aOoYf rnnlqrlad "pea., ErsPmp rnn Wen, ft WO Whir *0be taken 001W OW German GLPMqmlorrg Prenrsnrns and ft GLP Csrahcate rwaanes wwaea Untersc"t, Datum

I Signature, Date



### .. ... .~~

.. .. ..,M . .~.o~t .LL~o~- INarmund FuMran der rararlwoakMn Perm r narns aM Nndbn ar mPSna"'a Parson! Dr.-Ing. Karl-Hainz Rother - Prisident - l .andesamt fiir Umwefl, Wasserwictschaft und Gewerbeaufsicht

KaiserFriedrich-Stra(!e 7 55118 Mainz

. .. . . . . . .. . . . . . ., . . . . ., . . . . Wams WW

Adrpsa der GIP- . . . . . . . . . . . . . . . . . . . . . Name and aCras of the GLPrdomroriny Auw e~nql r Larnknan8 Illr Ltnrnclt . W,t,%crattt'hnll unit Gcacrhcaa(sicht 

# 0 Datum der Inspektion I Date of inspection (Tag. MonstJahr I day .monlh year) 27. und 28. November 2006 Dfs penanna PNhinrkMvq tfallnoal troh Mmnabonabn OLPd7WrwldrutqawMINM und, whd rnpanM6ip aW Ernhahunpda GLP.4runafttt utrsrwaahr AW der Gtnndtaq. dn tn+PSkfiarwenahN . *Yd hfnmit batlript. does In dinar PeWswfeMueq Cat Own qaean n~ bn PnrrwrpM udet EeMllunq der GLPGrw~=W

drpahrhrr warMn bOn»n. LM WhOrdIiMS (Ahptifwg der EInhaNuny der GlP-GrunOSllin duroh do PrUhknkMUnq at so recM :Mp tu bManusaan . date db FdpskupHnbn sWaAensvisr Jahn naen darn aaqlnn deroq tnspakhan srrirMWan karm 06n dwtM Anlqq whd die PqiralnnaMunp naeh I10trvr der Fnsr Bus dam dauMOhen GlP-UMmaehunqsprcqrane qMpmmsn und daNWk aesaMinytunp vwrlwA rhre OOKtqhtil

page 24 of 37Final Report of LAUS GmbH Auf der Schafwaide 20, D-67489 Kirrweiler Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info 14 ANNEX 2: DATA OF THE FIRST EXPERIMENT laus .de No . : AB07120702G803 Date: 02. May 2008

14 .1 Determination of Titre Criterion : The determination of titre should give a number of at least 109 cells/mL,

correlating to 100 colonies/plate after dilution. Table 14.1-a Titre Values (colonies per plate) Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Re I . 1 -S9 1001 1001 1001 1001 1001 Re I . 2( -S9

) 1001 1001 1001 .1001 1001 Re ( . 3( +S9

) 1001 1001 1001 1001 1001 Re I . 4 ( +S9 ) 1001 1001 1001 1001 1001 Mean 1001 1001 1001 1001 1001 sd 0 _ 0 0 0 0 ~ Assessment ~ ok~ ok ok ok ok In this table, "> 1000" is represented by "1001" . 14 .2 Sterility Control Criterion : At the maximum, one colony per plate may grow . Table 14.2-a Sterility (colonies per plate) H20 DMSO Re I . 1 0 0 Re I . 2 0 0 Re I . 3 0 0 Re I . 4 0 0 ~ Assessment ~ ok ok

page 25 of 37Final Report of LAUS GmbH Auf der Schatweide 20, D-67489 Kimveiler No. : AB07120702G803 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@taus.de I Date: 02. May 2008 14.3 Spontaneous Revertants Criterion : Revertants should lie for TA 97a between 90 and 180, for TA 98 between 30 and 50, for TA 100 between 120 and 200, for TA 102 between 240 and 320 and for TA 1535 between 80 and 300 colonies per plate (values by Prof. Ames) . The determined values were within the normal range of the laboratory. 14.3.1 Water Table 14.3-a Spontaneous Revertants H20 (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 1 02 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 

### _ +S9 -S9 +S9 Re I . 1 61 90 16 6 203 128 118 211 9 16 Re I . 2 131 94 7 8 230 190 198 266 8 10 Re I . 3 163 98 11 8 167 160 _ 188 131 10 10 Re I . 4 167 100 4 14 160 167 181 111 11 10 Mean 131 96 10 9 190 161 171 180 10 12 sd ~ 49.1 ~ 4.4 5.2 3.5 32 .6 25.6 36.21 71 .91 1 .3 1 3.0 14.3 .2 DMSO Table 14.3-b Spontaneous Revertants DMSO (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re (. 1 153 163 15 8 189 194 190 184 17 25 Re ( .2 150 116 9 10 138 196 177 . 179 15 33 Re I . 3 78 97 11 10 197 153 178 183 13 10 Re 1 .4 81 67 12 8 196 183 166 143 10 17 Mean 116 111 12 9 180 182 178 172 14 21 sd~ 41 .6 ~ 40.3~ 2.5 1,2 28.2 19 .8 9.8 196 30 99

page 26 of 37Final Report of LAUS GmbH I No. : AB07120702G803 Auf der Schatweide 20, D-67489 Kirrweiler Tel . ++49-6321-96299-0. Fax 96299-29, e-mail : infoQlaus.de 14 .4 Positive Controls Without metabolic activation : 4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 80 Fl,g/plate Sodiumazide (Na-azide) in deionised water, 6 pg/plate With metabolic activation : 2-Amino anthracene (2-AA) in DIVISO, 3 iag/plate Benzo-a-pyrene (BaP) in DMSO, 40 Etg/plate Table 14 .4-a Diagnostic Mutagenes (colonies per plate) Date: 02. May 2008



### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction 

### -S9

. +S9 -S9 +S9 -So +S9 -S9 +S9 -S9 +S9 Reference NPD 2-AA NPD BaP Naazide 2-AA NPD 2-AA Naazide 2-AA Re 1 .1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Re 1 .2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Re 1 .3 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Re L.4 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 sd 0 0 0 0 0 0 0 0 0 0 

### f(l ) 8.63 9 .02 83.42 111 .2 5.27 5.50 5 .62 ' 6.82 100,1 47.67 ~ Rev. abs. ~ 885 ~ 890 989 992 811 819 823 829 991 980' In this table, "> 1000" is represented by "1001" .

page 27 of 37Final Report of LAUS GmbH No. : AB07120702G803 Auf der Schafweide 20, D-67489 Kirrweiler Tel . ++49-G321-96299-0, Fax 96299-29, e-mail : info@laus .de Date: 02. May 2008 14.5 Test Item NHS (Sodiumhydroxymethanesulfinate) 14.5.1 Mutagenicity Test Table 14.5-a Concentration 5047 Nglplate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 111 114 13 5 169 152 53 165 8 14 Re I . 2 67 111 5 8 184 151 100 123 16 13 Re I . 3 182 127 15 10 194 126 104 140 7 15 Re I .4 117 88 14 6 109 129 70 198 10 20 Mean 119 110 12 7 164 140 82 157 10 16 sd 47 16 5 2 38 14 24 33 4 3 f I 0.91 1 .15 1 .20 0 .78 0.86 0.87 0.48 0.87 1 .00 1 .33 ~ Rev. abs. 1 -12 14 2 -2 -26 -21 -89 -23 0 4 Table 14.5-b Concentration 1514 Ng/plate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re L.1 131 144 11 5 182 123 189 196 10 7 Re ( . 2 110 166 9 13 197 218 194 127 10 7 Re I . 3 103 124 13 6 221 184 _ 111 159 10 8 Re ( .4 76 115 10 2 129 128 113 61 9 10 Mean 105 137 11 7 182 163 152 136 10 8 ad 23 23 2 5 39 46 46 57 1 1 f( l ) 0.80 1 .43 1 .10 0.78 0.96 1 .01 0.89 0.76 1 .00 0 .67 ~ Rev. abs. 1 -26~ 41 1 -2 -8 2 -19 -44 0 -4 Table 14.5-c Concentration 505 pglplate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 166 110 9 6 180 174 116 109 8 10 Re I . 2 156 157 7 10 168 129 139 92 9 10 Re I . 3 94 69 13 7 128 147 111 139 7 10 Re I .4 129 153 9 7 194 121 135 126 10 9 Mean 136 122 10 8 168 143 125 117 9 10 sd 32 41 3 2 28 24 14 20 1 1 f(1 ) 1 .04 1 .27 1 .00 0 .89 0 .88 _ 0 .89 0 .73 0,65 0

.90 0 .83 Rev. abs . ~ 5

J_ 26 1 01 -1 -22 -18 -46 -63 -1 -2

page 28 of 37Table 14 .5-d Concentration 151 Nglplate (colonies per plate)



### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re 1 .1 157 117 11 11 122 223 106 108 12 10 Re I . 2 67 112 7 12 95 163 145 131 17 17 Re I . 3 85 152 11 8 124 180 125 139 5 8 Re 1 .4 81 110 9 9 195 138 144 150 10 9 Mean 98 123 10 10 134 176 130 132 11 11 sd 40 20 2 2 43 36 18 18 5 4 f

### ( I

### ) 0.75 1 .28 1 .00 1 .11 0.71 1 .09 0.76 0.73 1 .10 0 .92 Rev . abs. -33 27 0 1 -56 15 -41 -48 1 -1 Table 14.5-e Concentration 51 pglplate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 75 145 12 6 210 138 203 126 10 7 Re I . 2 133 162 6 6 220 193 186 123 11 8 Re I . 3 139 110 14 5 191 188 130 156 17 1 0 Re ( .4 128 140 17 5 149 150 162 190 18 9 Mean 119 139 12 6 193 167 170 149 14 9 sd 30 22 5 1 31 27 32 31 4 1 

### f(l ) 0.91 1 .45 1 .20 0.67 1 .02 1 .04 0 .99 0.83 1 .40 0.75 Rev. abs. -12 43 2 -3 3 6 -1 -31 4 -3

page 29 of 37Final Report of i_AUS GmbH No . : AB07120702G803 Auf der Schatweide 20, D-67489 Kimveilet Tel . ++49-8321-96299-0, Fax 96299-29, e-mail : infoClaus.de Date: 02. May 2008 15 ANNEX 3

: DATA OF THE SECOND EXPERIMENT 15.1 Determination of Titre Criterion: The determination of titre should give a number of at least 109 cells/mL,

correlating to 100 colonies/plate after dilution . Table 15.1-a Titre Values (colonies per plate) Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Re ( .1 ( -S9

) 1001 1001 1001 ~1001 1001 Re (. 2( -S9

) 1001 1001 1001 1001 1001 Re I . 3( +S9

) 1001 1001 1001 1001 1001 Re l . 4 ( +S9 ) 1001 1001 1001 1001 1001 Mean 1001 1001 1001 1001 1001 sd 0 0 0 0 0 Assessment ok ok ok ok ok In this table, "> 1000" is represented by "1001 " . 15.2 Sterility Control Criterion: At the maximum, one colony per plate may grow . Table 15 .2-a Sterility (colonies per plate) H 20 

### DMSO Re I . 1 0 0 Re I . 2 0 0 Re I . 3 0 0 Re I . 4 0 0 Assessment ok ok

page 30 of 37Final Report of LAUS GmbH No. : AB07120702G803 Auf der Schafweide 20, D~67489 ICfnweiler Tel . ++49-6321-98299-0, Fax 96299-29, e-mail : infoOlaus.de Date: 02 . May 2008 15.3 Spontaneous Revertants Criterion: Revertants should lie for TA 97a between 90 and 180, for TA 98 between 30 and 50, for TA 100 between 120 and 200, for TA 102 between 240 and 320 and for TA 1535 between 80and 300 colonies per plate (values by Prof. Ames) . The determined values were within the normal range of the laboratory. 15.3 .1 Water Table 15.3-a Spontaneous Revertants HZO (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 ! Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 121 171 15 15 117 104 150 212 11 15 Re I . 2 112 194 10 9 110 131 154 280 - 6 8 Re I . 3 85 176 9 15 108 136 135 231 9 13 Re 1 .4 168 160 12 2 128 126 214 229 15 9 Mean 122 175 12 10 116 124 163 238 10 11 ad 34.6 14.2 2.6 6-2 9.0 14.1 34 .8 29.3 3.8 3,3 15.3 .2 DMS(J Table 15.3-b Spontaneous Revertants DMSO (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 141 126 16 7 154 170 129 252 13 15 Re I . 2 104 183 10 11 110 186 206 121 15 9 Re 1 .3 154 144 25 10 122 108 236 258 12 14 Re I . 4 149 161 14 11 213 126 197 200 13 8 Mean 137 154 16 10 150 148 192 208 13 12 sd 22.6 24

.3 6 .3 1 .9 46.1 36.6 45.2 63.4 1 .3 3.5

page 31 of 3715.4 Positive Controls

Without metabolic activation : 4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 80gg/plate Sodiumazide (Na-azide) in deionised water, 6 Etg/plate With metabolic activation: 2-Amino anthracene (2-AA) in DMSO, 3 Ng/plate Benzo-a-pyrene (BaP) in DMSO, 40 pg/plate Table 15.4-a Diagnostic Mutagenes (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Reference NPD 2-AA NPD BaP Naazide 2-AA NPD 2-AA Naazide 2-AA Re 1 .1 1001 1001 1001 1001 _1001 1001 1001 1001 1001 1001 Re l .2 1001 1001 1001 1001 1001 1001 1001 1001 1001 , 1001 Re 1 .3 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Re 1 .4 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 sd 0 0 0 0 0 0 0 ~ 0 0 0 f I 7.31 6.50 62.56 100.1 8.63 6.76 5.21 4 .81 100.1 83.42 ~Rev. abs. ( 864~ 8471 985~ 991 885 853 809 793 991 989 In this table, "> 1000" is represented by "1001" .

page 32 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-87489 Kirrvveiler No . : ABU7120702G803 Tel . ++49-6321-98299-0, Fax 98299-29, e-mail : infa@faus .de I Date: 02. May 2008 15.5 Test Item NHS (Sodium hydroxymethanesuifinate) 15.5.1 Mutagenicity Test Table 15 .5-a Concentration 5031 Nglplate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 S9 +S9 Re I . 1 136 148 8 11 141 101 198 229 13 13 Re I . 2 119 168 8 18 156 115 211 234 17 8 Re I . 3 138 169 12 8 127 213 215 296 14 13 Re I . 4 110 131 10 12 149 158 117 199 11 8 Mean 126 154 10 12 143 147 185 240 14 11 sd 14 18 2 4 12 50 46 41 3 3 f(l ) 1 .03 0.88 0 .83 1 .20 1 .23 1 .19 1 .13 1 .01 1 .40 1 .00 L Rev. abs . I 41 .21 1 .2 1 2 1 - 27 23 22 2 4 0 Table 15.5-b Concentration 2516 Nglplate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 93 82 9 10 124 115 234 180 7 17 Re 1 .2 112 86 10 11 145 196 282 113 10 16 Re ( . 3 131 100 10 8 140 199 212 242 11 15 Re 1 .4 70 136 19 11 _ 112 100 217 215 8 12' Mean 102 101 12 10 130 153 236 188 9 15 sd 26 25 5 1 16 52 32 56 2 2 f l 0.84 0 .58 1 .00 1 .00 1 .12 1 .23 1,45 0.79 0.90 1 .36 ~ Rev. abs. -20 -74 0 0 14 29 73 -50 -1 4', Table 15.5-c Concentration 1258 Nglplate (colonies per plate) 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Induction -S9 +S9 -S9 

### _ +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 203 134 8 4 210 177 222 197 11 13 Re I . 2 89 90 9 10 208 113 218 197 9 13 Re I . 3 175 170 16 15 202 172 225 . 120 12 13 Re ( .4 94 122 11 12 192 143 247 158 13 13 Mean 140 129 11 10 203 151 228 168 11 13 ad 57 33 4 5 8 30 13 37 2 0 f I 1 .15 0.74 0.92 1 .00 1 .75 1 .22 1 .40 0 .71 1 .10 1 .18 ~ Rev . abs. ~ 18 1 -46~ -1 ~ 0 ~ 87 27 65 -70 1 2~

page 33 of 37Final Report of LAUS GmbH Auf der Schafweide 20, D-67489 Kirrweiler No. : AB07120702G803 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : infoQlaus .de I Date: 02. May 2008 16 ANNEX 4

: DATA OF THE PRE-TEST (CYTOTOXICITY) As the pre-test was not performed under GLP-conditions, only the values for the titre and the toxicity control are stated in this report . The toxicity of the following concentrations were tested : 5017 and 1505 lag/plate

. Per strain, four plates were incubated with the corresponding dose of the test item on maximal soft agar . The treatments for the confirmation of the genotype, the sterility control and the determi- nation of the titre didn't showany inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation . 16.1 Experimental Parameters Date of treatment January 09th 2008 Concentrations tested Incubation time Incubation temperature Tester strains Method 16.2 Determination of Titre Criterion: Table 16.2-a 5017 / 1505 lag/plate 48 hours 37 C TA97a, TA98, TA100, TA102, TA1535 Plate incorporation method The determination of titre should give a number of at least 109 ceIIs/mL, correlating to 100 colonies/plate after dilution. Titre Values (colonies per plate) Strain TA 97a TA 98 TA 100 TA 102 TA 1536 Re I . 1 -S9 1001 1001 1001 1001 1001 Re (. 2 (-S9 ) 1001 1001 1001 1001 1001 Re I . 3 ( +S9 ) 1001 1001 1001 1001 1001 Re I . 4 ( +S9 ) 1001 1001 1001 '1001 1001 Mean 1001 1001 1001 1001 1001 8d 0 0 0 0 0 Assessment ok ok ok ok ok In this table, "> 1000" is represented by "1001" .

page 34 of 37Final Report of LAUS GmbH Auf der Schaiweide 20, D-67489 Kirrweiier Tel . ++q9-6321-96299-0, Fax 96299-29, e-mail : info@iaus.de No. : AB07120702G803 Date: 02. May 2008 16.3 Toxicity Control The test itemis considered non-toxic, if the quotient titreltox is below2 . Table 16,3-a 5017 pglplate on maximal-soft-agar (colonies per plate) Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Re (. 1 -S9 1001 1001 1001 1001 1001 Re I . 2 ( -S9 ) 1001 1001 1001 1001 1001 Re I . 3 +S9 1001 1001 1001 .1001 1001 Rep] . 4 +S9 1001 1001 1001 1001 1001 Mean 1001 1001 1001 1001 1001 sd 0.0 __ 0.0 0 .0 0.0 0 .0 ~ TitrelTox ~ 1 .00 ~ 1 .00 1 .00 1 .00 1 .00 Table 16.3-b 1505 Nglplate on maximal-soft-agar (colonies per plate) Strain TA 97a TA 98 100 TA 102 TA 1535 Re I . 1 -S9 1001 1001 1001 1001 1001 Re 1. 2 ( -S9 ) 1001 1001 1001 1001 1001 Re I . 3( +S9

) 1001 1001 1001 1001 1001 Re I . 4 +S9 1001 1001 1001 1001 1001 Mean 1001 1001 1001 1001 1001~ ad 0.0 0 .0 0.0 0.0 0.0 ~ TitrelTox ~ 1 .00 ~ 1 .00 1 .00 1 .00 1 .00 16.4 Assessment On the base of these results, the first experiment was performed with 5000 Ng/plate as maximumdose for all strains .

page 35 of 37Final Report of LAUS GmbH Auf der schafweide 20, D-67489 Kirnaeiler No. : AB07120702Ga03 Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus.de I Date: 02. May 2008 17 ANNEX 5

: COMPARISON WITH HISTORICAL DATA In the following table, the mean of the spontaneous revertants and positive controls of all performed experiments in the year 2007 is stated (Number of experiments : 20) in comparison with the experiments performed within this study. Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Induction I - S9 +S9 - S9 +S9 - S9 + S9 - S9 +S9 - S9 + S9

Mean 127 143 24 20 144 151 181 208 13 17 Min 81 79 11 12 110 131 134 180 6 10 H20 Max 160 179 53 37 177 180 242 255 26 21

Ex 1 131 96 10 9 190 161 171 180 10 12 Ex 2 122 175 12 10 116 124 163 238 10 11 Mean 134 130 14 15 146 142 186 179 13 13 Min 94 TA 98 8 6 105 106 120 117 8 5 DMSO Max 190 182 47 53 204 179 267 277 21 21

Ex 1 116 111 12 9 180 182 178 172 14 21 Ex 2 137 154 16 10 150 148 192 208 13 12 Mean 1001 971 1001 717 1001 1001 1001 1001 1001 908 Min 1001 467 1001 150 1001 1001 1001 1001 1001 139 Pos. Contr, Max 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001

Ex 1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 Ex 2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001

page 36 of 37Final Report of LAUS GmbH Auf der Schafweide 20. D-67489 Kirrweiier Tel . ++49-6321-96299-0. Fax 96299-29, e-mail : Info 18 ANNEX 6

: GENOTYPE CONFIRMATION Date of Performance: January 09th 2008 18.1 Histidine Requirement iaus.de No. : AB07120702G803 Date: 02. May 2008

Criterion : On the biotin plates, no growth should occur, whereas on the histidine-

biotin plates, the strains should grow well . Found : Growth on histidine-biotin plates, no growth on biotin plates. Assessment : okay 18.2 Ampicillin-(Tetracyclinee)-Resist

. (pKM101, pAQ1) Criterion : After incubation for 12 hours, good growth should be observed on the am-

picillin plates for all strains except TA1535. On the ampicillin-tetracycline plates, only TA102 should grow . Found: TA97a, TA 98, TA 100, TA 102 grow in the presence of ampicillin, only

TA102 with ampicillin and tetracyclinee, TA 1535 doesn't growanywhere. Assessment : okay 18.3 UV-Sensitivity (uvrB) Criterion : On the irradiated side of the plates, only TA 102 should grow, whereas on

the side, which was protected from light, all strains should grow . Found : all strains grow on the non-irradiated half, only TA102 on the irradiated

one. 

### Assessment : okay. 18.4 Crystal Violet Sensitivity (deep roughirfa) Criterion: The zone of inhibition has a diameter of at least 14 mm . Found : Table 18.4-a Inhibition in mm 

### Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Re l . 1 15 14 15 16 15 Re I . 2 15 14 16 16 15 Mean 15 14 15 .5 16 15 Assessment : okay.

page 37 of 37Endpoint study record: Genetic toxicity in vitro .001 file:///C:/Dokumenke >,nd EinstellungenPil/Desktop/index.htrnl

Printing Date 2011-03-07 13:11 :33 CET Restriction of specific regulatory purposes EU : BPD, EU: PPP, EU: REACH, CA : CEPA, CA: PCPA, JP: CSCL, OECD: HPVC, US: EPA HPVC

. US: FIFRA, US : TSCA, other

. Confidentiality CBI, IP, no PA Owner Sodium hydroxymettmnesulphinate / sodiumhydroxymelhanesulphinate /sodiumhydroxymeMianesulfinate / 149-44-0 /

Briiggemannehemical, L. Bruggemann KG / Heilbronn/Germany Legal entity Biiiggemannehemical L. Briiggemann KG / Heilbronn /Gemiany ownerEndpoint study record: Genetic toxicity in vitro.001 fi1e:///C :/Dokumente ind Einstellungen&/tesktop/indexhtml 

### Endpoint study record: Genetic toxicitv in vitro.001 UUID IUC5-9bbede48-512a-4ace-a523-80320135dedd Dossier UUID 0 Author UL / BruggemannChemical L. Briiggemann KG / Heilbronn / Germany Date 2011-03-07 13:10:57 CET Remarks Administrative Data EU: REACH Purpose flag key study, robust study summary ; usedfor classification ; used for MSDS Study rosuRtype experimental result Study period January 2008 - May 2008 Reliability 1 (reliablewithoutrestriction) Rationale for GLPguideline study . Planning and conductof the studywere based on the OECDguideline "Genetic Tobcology . Salmonella reliability incl . typNmurium, Reverse MutationAssay", OECD No. 471 OECD Guidelines forttie Testing of Chemicals Bacterial Reverse deficiencies MutationTest' Part 471, adopted July 21 st 1997 EU-Guideline 67/548 EWG, last changed by 29th amendment AnnexV,

Method B.13/14 "Mulagenicity- Salmonella typhimurium, reverse mutation assay, adopted May 191h, 2000 Data source Reference

Data access Reference type Author Year Title Bibliographic source Testing

laboratory Report no. Ownercompany Company study no. Report

date study Paulus, 2008 Determination ofthe LAUS AB07120702G803 BruggemamChemical, 2008_05_02_01 2008-05-20 report Jbrg mulagertic potential of GmbH; L.Bruggemann KG NHS (Sodium Kimveiler, hydroxymethanesuffinate) Germany with the Bacterial Reverse Mutation Test data submitter is data owner Data protection claimed yes, butwillingto share Materials and methods Type of genotoxicity gene mutation Type of study bactedal reverse mutationassay (e.g.Ames best) Test guideline Qualifier Guideline Deviations accordingto OECD Guideline471 (Bacterial Reverse Mutation Assay) (OECD471) no I accor ding to EU Method 8

.1 3/14 (Mu

lag enicity- R

ev erse Mutati on Test Using Bacteria) no GLPcompliance yes (incl . certificate) Test materials Identity of test material same as for substance defined in section 1 (if not read-across) yes Confidential details on test material Lot no. :07041205 Method Target gene his-of Salmonella typhimiriumLT2 Strains : TA 1535, TA 97a, TA 98, TA 100and TA 102 Species/strain Specieslstraln S. typhimurium TA 98 Additional other

. hisD3052, uvr8, pKM

101, rfa strain characteristicsEnapoint study record: Genetic toxicity in vitro.001 file:///C:/Dokwnente wid Einstellunw~genul/Desktop/indexhtml

Metabolic with and without activatbn Metabolic Rat liverS9 metabolicactivation activation system Specleslstrain S. typhimurium, other . TA97a Additional other

. hisD6610, uvrB, pKM

101, rfa strain characteristics Metabolic with and without activation Metabolic Ratliver S9 metabolic activation activation system Specles/straln S

. typhimurium

TA 100 Additional other

. hisG46, uorB, pKM

101, rfa strain characteristics Metabolic with and without activation Metabolic Ratlirer S9 metabolic activation acfhratbn system Specles/strain S. typhimurium TA 102 Additional other

. hisG428, pKM

101, rfa strain characteristics Metabolic with andwithout activation Metabolic Rat liwr S9 metabolic activation activation system Specleslstrain S. typhimurium TA 1535 Additional other

. HisG46 strain characteristics Metabolic with and without activation Metabolic Rat liverS9 metabolicactivation activation system Test concentrations Experiment I (plats incorporation mettiod) : conc : 5047 / 1514 / 505/151 / 51 Ng/plats Experiment II (pre-incubation method) : conc. : 5031 / 2516 / 1258 Ng/plate vehicle -vehide(sysolvenE(s) used: [water] - Justification forchoice of sohenf/vehicle : test itemwas completely soluble inwater, and waterdoes not ham anyefects on theviability of the bacteria or the number of spontaneous reoertanFs . Controls Solvent/ yes (water) vehicle controls Positive yes (TA 97a, TA 98 and TA 102) controls Positive other

. 4Nitno-1,2-pherrylene diamine (80Ng/plate) control substance Remarks without metabolic activation system Solvent/ yes (water) vehicle controls Positive yes (rA 100and TA 1535) controls Positive sodiumaade (8 Ng/plate) control substanceEndpoint study record: Genetic toxicity in vitro.001 file:///C:/Dokuniente und Einstellungenul/Deslctop/index.html

Rernarks withoutrnstabolicadivation system Solventl yes (water) vehicle controls Positive yes (TA 98) controls Positive benao(a)pyrore (40Nglplate) control substance Remarks with metabolicactivation system Negative yes controls Solvent/ nodafa vehicle controls True yes negative controls Positive yes (TA 97a, TA 100, TA 102and TA 1535) controls Positive other

. 2-AminoardFuacene (3 Nglplate) control substance Remarks with metabolic activation system Details on test systemand conditions METHOD OF APPLICATION : in agar (plate incorporation) - Preincubation period : 12 hours . PLATE INCORPORATION METHOD (First Experiment) : - Per strain and dose, four plates with and four plates withoutS9 mixwere used. 10 mL of the test solution of to appropriate concentrationwere membrane flllratsd into sterile vessels . Topagar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-solution 0 .5 mMol per 100 mL basis was added and the bottle was placed intowater bath at 45 °C. 0 .1 mL of theappropriate solution of thetest itemwere giveninto a sterile tube. After mixing with 0 .1 mL overnight culture of the respective strain and 0 .5 mL phosphate buffer (onlyfor treatmerb withoutS9) or 0 .5 mL S9 mix, 2 ml . Top-Agar were added. The mixture was gently eortexed, then poured on a minimal glucose plate anddistributed erenly,using a Drigalski Spatula . The plates were closed, coveredwith brown paperand left to hardenfora few minutes, then inverted and placed in the dark incubatorat 37 °C. PRE-INCUBATION METHOD (second experiment) : - Perstain and dose,four plates with and four plates withoutS9 mixwere used. 10 mL of the test solution of the appropriate concentrationwere membrane filirated into sterile vessels . Top agar basis was melEed in a microwave oven, aftermelting, 10 ml . of Itistidinebiotin- solution 0 .5 mMol per 100mL basiswas added and the bottle was placed in the water bath at 45 °C. 0 .1 mL of the appropriate solution of the test itemwere given into a sterile tube . After mixing with 0 .1 ml . overnight culture of the respective strain, 0 .5 mL phosphate buffer (only for treatments withoutS9) or 0 .5 mL S9 mixwere added. The mixture was incubated in an incubation chamberat 37°C for20 minutes . During this time the vesselswere aerated through careful shaking

. Then 2 mL top agarwere added . The mixturewas oortexed gently, then poured on a minimal glucose plateand distributed eoerNy, using a Drigalski spatula . The plates were dosed, covered with brownpaperand leftto harden forafew

minutes, then inverted and placed in the dark incubator at 37 °C. NUMBEROF REPLICATIONS: Four replicates, withMthout S9, foreach solvent which was used in thetest Evaluatlon criteria Atest itemis considered to have muFagenic potential, if a significant, reproducible increase of revertantcolonies perplate (ncrease factor 2) in at least onestrain canbe observed . Aconcentration-related increase over the range tested can also be takenas a sign of mutagenicactioity . Statistics STATISTICAL METHDODS USED: The colonies were counted visually, the numbers were recorded .Aspreadsheetsoftware (Microsoft Excel®)was used to calculate mean values and standard deviations aswell as the increase factor of revsrtant induction. Results and discussions Test results Speciieslstrain other: TA 97a, TA98, TA 100, TA 102 and TA 1535 Metabolic with activation Test systam all strains/call types tested (First Experiment) Genotoxicity negative Cytotoxk:Uy no Vehicle yes controls valid Negative yes controls valid Positive yes controls validEndpoint Ar%

study record: Genetic toxicity in vitro .001 file :///C:/Dokumente und Einstellungenul/Ifesktop/index .html Species/strain other

. TA 97a, TA 98, TA 100, TA 102 and TA 1535 Metabolic without activation Test system all strains/csl types tested (First Experiment) Genotoxicity negative Cytotoxicily no Vehicle yes controls valid Negative controls valid yes Positive yes controls valid Specieslstrafn other

. TA 97a, TA 98,TA 100, TA 102and TA 1535 Metabolic with activation Test system all strains/cell types tested (Second Experiment) Genotoxicity negative Cytotoxiclly no Vehicle yes controls valid Negative controls valid Positive controls valid yes

yes Specieslstraln other

. TA 97a, TA 98, TA 100, TA 102and TA 1535 Metabolic without activation Test system all strainslcell types tested (Second Experiment) Genotoxicity negative Cytotoxicity no Vehicle yes controls valid Negative yes controls valid Positive yes controls valid Additional informatlon on resnlts OBSERVATION FIRST EXPERIMENT: " Thetreatments forthe confirmation of the genotype, the sterilitycontrol and thedeterminationof thetiterdid not show anyinconsistencies. " All positive controls (diagnostic mulagens) showed mulagenic effectswith and without metabolic activation

. " No signs of toxicity towards the tested strains could be observed : " The background lawn was visible ; the number of revertant colonies was not significantly reduced. " No significant increase of the number of reverlantcolonies in the treatments with and without metabolic activation could be observed . " No concenh-ation-related increase over thetested range was found. SECOND EXPERIMENT: " The treatments forthe confirmation of the genotype, the stsrilitycontrol and the determination of the titerdid notshow arry inconsistencies . " All positive controls showed mulageniceffectswith and without metabolic activation. " No signs of toxicity towards the tested strains could be observed: " The background lawn was visible; the number of reoertantcolonies was notsignificantly reduced. " No significant increase of the number of revertantcolonies in the treatments with and without metabolic activation was observed . " No concentration-related increase over the tested range was found. COMPARISON WITH HISTORICAL CONTROL DATA: The determined values forthe spontaneous revertards of the negative controls were in the normal range of thetest laboratory historical data of the laboratory (in the first and in the second experiment) . Applicant's summary and conclusion Interpretation of results negative with metabolic activation negativewithout metabolic activation Conclusions The test itemis considered to ben non-mufagenic in the bacterial reverse mutation test Executive sunriffiary First experimentEr~dpoint study record: Genetic toxicity in vitro.001 file:///C:/Dolamnente und EinstellungeNul/4D6eslctop/index .html Five concentrations of the test item, dissolved in deionised water (ranging from5047 to 51 ug/plate) were used. Five genetically manipulated strains of Salmonella typhimurium (TA97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test itemboth in the presence and in the absence of a metabolic activation system(S9) for 48 hours, using the plate incorporation method. None of the concentrations caused an increase in the number of rewertant colonies in the tested strains. The test itemdidn't showany mutagenic effects in the first experiment . The test itemdidn't show any cytotobcity towards the bacteria. The sterility control and the determination of the titer did not showany inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range . All positive controls showed mutagenic effects with and without metabolic activation. Second exoeriment To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging from

5031 to 1258 Ng/plate) and a modification in a study performance (pre-incubation method)

. The test itemdidn't showany mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titer did not showany inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range . All positive showed mutagenic effects with and without metabolic activation. Under the conditions of the test, the test itemdid not showmutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test itemis considered as "not mutagenic" under the conditions of the test .